论文标题

发现酵母质子泵,PMA1的扩散状态以及标记方法如何改变扩散行为

Uncovering diffusive states of the yeast proton pump, Pma1, and how labeling method can change diffusive behavior

论文作者

Bailey, Mary Lou P., Pratt, Susan E., Zhang, Yongdeng, Hinrichsen, Michael, Bewersdorf, Joerg, Regan, Lynne J., Mochrie, Simon G. J.

论文摘要

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently-labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1,然后绑定meos3.2。这两个单个粒子轨道的扩散率分布显着差异,表明标记方法可以是扩散行为的重要决定因素。我们还应用了扰动期望最大化(PEMV2)[物理审查E 94,052412(2016)],将轨迹分类为统计上最高数量的扩散状态。对于陷阱标记的PMA1和PMA1-MEOS3.2,PEMV2将轨道分为两个扩散状态:本质上是不动的状态和更移动的状态。但是,PMA1-MEOS3.2轨道的移动部分比陷阱标记的PMA1轨道的移动部分(0.5)小得多(0.1)。此外,PMA1-MEOS3.2移动状态的扩散率比陷阱标记的PMA1移动状态的扩散率小几倍。为了批判性地评估PEMV2的性能,我们比较了实验性PEMV2分级种群的扩散率和协方差分布与相应的理论分布,假设PMA1位移实现了高斯随机过程。陷阱标记的PMA1和PMA1-MEOS3.2的实验理论比较揭示了良好的一致性,并加强了PEMV2方法。

We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently-labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2. The diffusivity distributions of these two populations of single particle tracks differ significantly, demonstrating that labeling method can be an important determinant of diffusive behavior. We also applied perturbation expectation maximization (pEMv2) [Physical Review E 94, 052412 (2016)], which sorts trajectories into the statistically-optimum number of diffusive states. For both TRAP-labeled Pma1 and Pma1-mEos3.2, pEMv2 sorts the tracks into two diffusive states: an essentially immobile state and a more mobile state. However, the mobile fraction of Pma1-mEos3.2 tracks is much smaller (0.1) than the mobile fraction of TRAP-labeled Pma1 tracks (0.5). In addition, the diffusivity of Pma1-mEos3.2's mobile state is several times smaller than the diffusivity of TRAP-labeled Pma1's mobile state. To critically assess pEMv2's performance, we compare the diffusivity and covariance distributions of the experimental pEMv2-sorted populations to corresponding theoretical distributions, assuming that Pma1 displacements realize a Gaussian random process. The experiment-theory comparisons for both the TRAP-labeled Pma1 and Pma1-mEos3.2 reveal good agreement, bolstering the pEMv2 approach.

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